Beka Solomon

Modulation of Alzheimer’s β-amyloid neurotoxicity by site-directed single-chain antibody

A single-chain antibody was constructed from variable regions of heavy and light genes of the parental anti-beta-amyloid peptide IgM 508 antibody. This antibody exhibits anti-aggregating properties, leading to disaggregation of Alzheimer beta-amyloid (betaA) fibrils and prevents its toxic effect on cultured PC-12 cells. Sequencing of the small antibody, namely 508 (Fv), revealed that the V(L) domain contained a cysteine residue in the complementary determining region (CDR)3 (residue 96) which affects its solubility and stability. The cysteine codon was replaced using SOE PCR, and one of the mutants obtained, namely 508F(Fv) (containing phenylalanine instead of cysteine), showed an increased storage stability and higher affinity compared to the wild type. Antibody 508F(Fv) prevents the neurotoxicity of betaA (90% cell viability) and disrupts the fibril structure of beta-amyloid (62% decrease in ThT fluorescence). The ability of antibody 508F(Fv) to dissolve already-formed betaA fibrils makes it a good candidate for intracellular expression and modulation of APP processing as the first step towards the production of therapeutic protection molecules for Alzheimers disease treatment.

N-terminal EFRH sequence of Alzheimer's β-amyloid peptide represents the epitope of its anti-aggregating antibodies

Monoclonal antibodies 6C6 and 10D5 raised against the N-terminal of beta-amyloid peptide interfere with the formation of beta-amyloid and trigger reversal to its non-toxic components. The epitopes of these antibodies were localized employing a library composed of filamentous phage displaying random combinatorial hexapeptides. Among 44 positive phage-clones, selected from the library by both antibodies, 40 clones carried the consensus sequence EFRH. These EFRH phage-clones bind specifically mAbs 6C6 or 10D5 with an apparent binding constant of approximately 10(-9) M. The peptide EFRH inhibits binding of mAbs 6C6 or 10D5 to beta-amyloid peptide in affinities identical to those obtained. with the peptides corresponding to positions 1-9, 1-16 and 1-40 of beta-peptide. These findings confirm that the peptide EFRH which is located at positions 3-6 within beta-amyloid peptide represents the sequential epitope of mAbs 6C6 and 10D5.

Filamentous phage as vector-mediated antibody delivery to the brain

Early diagnosis of Alzheimers disease is prevented by lack of means to visualize and target β amyloid plaques in the brains of affected people. There are many methods of detecting amyloid plaques by staining postmortem brain tissue, but none are available for monitoring in living patients. We propose anti-β amyloid antibodies as a highly specific probe to monitor amyloid plaque formation in living patients. Intranasal administration of filamentous phage as delivery vector of anti-β amyloid antibody fragment into Alzheimers APP transgenic mice enables in vivo targeting of β amyloid plaques. The plaques were covisualized both by thioflavin-S and fluorescent-labeled antiphage antibodies in the olfactory bulb and the hippocampus region. The genetically engineered filamentous bacteriophage proved to be an efficient and nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors. The ability to image Aβ deposits in vivo would arguably provide the most useful diagnostic and monitoring test for early diagnosis of Alzheimers disease.

High affinity binding of monoclonal antibodies to the sequential epitope EFRH of β-amyloid peptide is essential for modulation of fibrillar aggregation

Monoclonal antibodies raised against the N-terminal of Alzheimers beta-amyloid peptide (betaAP) were found to modulate its fibrillar aggregation. While mAbs 6C6 and 10D5 inhibit the formation of beta-amyloid fibrils, trigger disaggregation and reversal to its non-toxic form, mAb 2H3 is devoid of these properties. MAb 2H3 binds the sequence DAEFRHD, corresponding to position 1-7 of the betaAP with high affinity (2 x 10(-9) M) similar to its binding with the whole betaAP. The EFRH peptide strongly inhibits binding of mAbs 6C6 and 10D5 to betaAP, whereas it inhibits weakly the interaction of 2H3 with betaAP. Low affinity binding of mAb 2H3 to EFRH might explain its failure in prevention of beta-amyloid formation.

Reduction of β-amyloid plaques in brain of transgenic mouse model of Alzheimer’s disease by EFRH-phage immunization

Antibodies to the epitope EFRH, representing residues 3-6 within the beta-amyloid (Abeta) sequence, were previously shown to affect the solubility and disaggregation of Abeta fibrils in vitro. Here, we describe a novel method of immunization, using as antigen the EFRH peptide displayed on the surface of the filamentous phage. The EFRH phage evoked effective auto-immune antibodies in amyloid precursor protein [V717I] (APP[V717I]) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimers disease (AD). The immunization provoked a considerable reduction in the number of Abeta amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-Abeta vaccine.

Intravenous immunoglobulin enhances the clearance of fibrillar amyloid-β peptide

Intravenous immunoglobulin (IVIg), a purified immunoglobulin fraction manufactured from the blood of healthy humans, is an FDA‐approved treatment for many immune and inflammatory diseases. Recent studies have demonstrated that IVIg therapy has several positive effects on patients with Alzheimers disease (AD). These include improving cognitive functions and lowering the level of soluble amyloid‐β peptide (AβP) in the brain. Nonetheless, the mechanism by which IVIg mediates the clearance of AβP from the AD brain currently remains unknown. In this study we investigated the molecular basis for the direct and indirect effects of IVIg on AβP clearance using the BV‐2 cellular microglia line. Specifically, we show that IVIg dissolves preformed AβP fibrils in vitro. Moreover, IVIg increases cellular tolerance to AβP, enhances microglial migration toward AβP deposits, and mediates phagocytosis of AβP. Thus, several mechanisms can be considered when examining the effects of IVIg. Our work supports the hypothesis that IVIg interferes by more than one mechanism in clearing AβP from the brains of Alzheimers patients.

EFRH-phage immunization of Alzheimer's disease animal model improves behavioral performance in Morris water maze trials.

We have developed an immunization procedure for the production of effective anti-β-amyloid (anti-Aβ) antibodies, using filamentous phage displaying only 4 amino acids. The EFRH sequence, encompassing amino acids 3–6 of the 42 residues of Aβ peptide, was found previously to be the main regulatory site for amyloid modulation and the epitope of anti-aggregating antibodies. Engineered filamentous phage enable the display of various numbers of EFRH copies on the phage and serve as potent carriers of antigens. In the present study we have found that phage displaying high EFRH copy number are effective in eliciting humoral response against the EFRH sequence, which in turn relieves the amyloid burden in the brains of amyloid precursor protein Tg mice and improves their ability to perform cognitive tasks.

VIP and peptides related to activity-dependent neurotrophic factor protect PC12 cells against oxidative stress

Oxidative stress is a common associative mechanism that is part of the pathogenesis of many neurodegenerative diseases. Vasoactive intestinal peptide (VIP) is a principal neuropeptide associated with normal development and aging. We have previously reported that VIP induced the secretion of proteins from glial cells, including the novel survival-promoter: activity-dependent neurotrophic factor (ADNF). ADNF-9, a nine amino acid peptide derived from ADNF, protects neurons from death caused by various toxins. In the present study, we examined the neuroprotective effect of VIP against oxidative stress in a pheochromocytoma cell line (PC12). In addition, a lipophilic derivative of VIP, Stearyl-Nle17-VIP (SNV), and two femtomolar-acting peptides: ADNF-9 and a 70% homologous peptide to ADNF-9, NAP were tested as well. PC12 cells were treated with 100 µM H2O2 for 24 h resulting in a reduction in cell survival to 35–50% as compared to controls. Addition of VIP or SNV prior and during the exposure to 100 µM H2O2 increased cell survival to 80–90% of control values. Culture treatment with ADNF-9 or NAP in the presence of 100 µM H2O2 increased cell survival to 75–80% of control values. Messenger RNA expression analysis revealed that incubation with VIP resulted in a twofold increase in VIP mRNA, whereas NAP treatment did not cause any change in VIP expression, implicating different mechanisms of action. Furthermore, addition of an ADNF-9 antibody prevented the ability of VIP to protect against oxidative stress, suggesting that VIP protection is partially mediated via an ADNF-like protein.

Generation of auto-antibodies towards Alzheimer's disease vaccination.

We developed a novel procedure to evoke anti-aggregating β-amyloid (Aβ) antibodies, using filamentous phages displaying only four amino acids EFRH of the β-amyloid peptide (AβP). This epitope was found to be the main regulatory site for fibril formation. For the first time, effective auto-antibodies have been obtained in guinea-pigs, which exhibit human identity in the AβP. Immunization through a phage-carrying epitope was found to be long-lasting, and no toxic effect caused by autoimmune response was detected in the challenged animal sections. These antibodies performed similarly to site-directed monoclonal antibodies and to antibodies raised against fibrillar Aβ in disaggregation of plaques, and may serve as the basis for developing an anti-Aβ vaccine.

Inhibitory effect of monoclonal antibodies on Alzheimer's β-amyloid peptide aggregation

In vitro aggregation of β-amyloid peptide induced by incubating the peptide for 3 h at 37°C, was investigated via immunocomplexation with a panel of monoclonal anti-bodies raised against various β-amyloid fragments Some of these monoclonal antibodies prevented the formation of β-amyloid and this effect may be related to the localization of the antibody binding sites. Monoclonal antibodies 6C6 and 10D5, and AMY-33, which recognize epitopes spanning the amino acid residues 1–28 of β-amyloid peptide, inhibited its aggregation by 40%–90% when compared with aggregation occurring in the absence of the respective antibodies. The antibodies, 2H3 and 1C2, directed to the regions comprisingpeptides 1–12 and 13–28, respectively, had a considerably low effect. Identifying the “aggregating epitopes” as sequences that are related to the sites where protein aggregation is initiated and preparing monoclonal antibodies against these regions may facilitate the under-standing and prevention of protein aggregation processes....